title: AMPKα1-sensitivity of Orai1 and Ca(2+) entry in T - lymphocytes.
abstract: OBJECTIVE
  T-lymphocyte activation and function critically depends on Ca(2+) signaling, which is regulated by store operated Ca(2+) entry (SOCE). Human and mouse T lymphocytes express AMP activated kinase AMPKα1, which is rapidly activated following elevation of cytosolic Ca(2+) concentration ([Ca(2+)]i) by treatment of the cells with Ca(2+) ionophore or following inhibition of endosomal Ca(2+) ATPase with thapsigargin. AMPK is further activated by triggering of the T cell antigen receptor (TCR). The present study explored whether AMPK influences Ca(2+) entry and Ca(2+)-sensitive regulation of T-lymphocyte function.
  
  
  METHODS
  T-lymphocytes were isolated and cultured from AMPKα1-deficient (ampk(-/-)) mice and from their wildtype (ampk(+/+)) littermates. The phenotype of the cells was analysed by flow cytometry, [Ca(2+)]i estimated from Fura-2 fluorescence, SOCE from increase of [Ca(2+)]i following thapsigargin treatment (1 µM), and cell function analysed by measuring cytokine secretion and western blotting.
  
  
  RESULTS
  Expression of surface markers in CD4(+) and CD8(+) T-cells were similar in ampk(-/-) and ampk(+/+) T-lymphocyte blasts. Moreover, total STIM1 protein abundance was similar in ampk(-/-) and ampk(+/+) T-lymphocyte blasts. However, Orai1 cell membrane protein abundance was significantly higher in ampk(-/-) than in ampk(+/+) T-lymphocyte blasts. SOCE and increase of [Ca(2+)]i following TCR activation by triggering TCR with anti-CD3 and cross-linking secondary antibody were both significantly more pronounced in ampk(-/-) than in ampk(+/+) T-lymphocyte blasts. The difference of Ca(2+) entry between ampk(-/-) and ampk(+/+) T-lymphocytes was abrogated by Orai1 inhibitor 2-aminoethoxydiphenyl borate (2-APB, 50 µM). Proliferation of unstimulated ampk(-/-) lymphocytes was higher than proliferation of ampk(+/+) T-lymphocytes, a difference reversed by Orai1 silencing.
  
  
  CONCLUSIONS
  AMPK downregulates Orai1 and thus SOCE in T-lymphocytes and thus participates in negative feed-back regulation of cytosolic Ca(2+) activity.
authors.affiliation: Department of Physiology, University of Tübingen, Tübingen, Germany.
authors.forename: Shefalee K
authors.initials: SK
authors.lastname: Bhavsar
authors.forename: Sebastian
authors.initials: S
authors.lastname: Schmidt
authors.forename: Diwakar
authors.initials: D
authors.lastname: Bobbala
authors.forename: Meerim K
authors.initials: MK
authors.lastname: Nurbaeva
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Hosseinzadeh
authors.forename: Katja
authors.initials: K
authors.lastname: Merches
authors.forename: Abul
authors.initials: A
authors.lastname: Fajol
authors.forename: Jan
authors.initials: J
authors.lastname: Wilmes
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001896:Boron Compounds / Q000494:pharmacology; D017252:CD3 Complex / Q000276:immunology / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D002478:Cells, Cultured; D016257:Fura-2 / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D000071740:ORAI1 Protein; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D000071737:Stromal Interaction Molecule 1; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D000906:Antibodies; D001896:Boron Compounds; D017252:CD3 Complex; D015220:Calcium Channels; D008562:Membrane Glycoproteins; D000071740:ORAI1 Protein; C520430:Orai1 protein, mouse; D034741:RNA, Small Interfering; D011948:Receptors, Antigen, T-Cell; C100757:Stim1 protein, mouse; D000071737:Stromal Interaction Molecule 1; D019284:Thapsigargin; C109986:2-aminoethoxydiphenyl borate; C548179:Prkaa1 protein, mouse; D055372:AMP-Activated Protein Kinases; D002118:Calcium; D016257:Fura-2